High density array fabrication and readout method for a fiber optic biosensor

ABSTRACT

The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its &#34;sensor end&#34; biological &#34;binding partners&#34; (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor.

This invention was made with the Government support under Grant No. CA45919, awarded by the National Institute of Health and under Grant No.DE-AC0376SF0098, awarded by the Department of Energy. The Government hascertain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Divisional of U.S. Ser. No. 08/448,043, filed on May 23, 1995,now U.S. Pat. No. 5,690,894, which is incorporated herein by referencefor all purposes.

FIELD OF THE INVENTION

This invention relates to the fabrication and use of biosensorscomprising biological "binding partners" (molecules that specificallybind other molecules to form a binding complex such as antibody-antigen,lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.)linked to optical fibers. Specifically, batches of optical fibers aremass processed with the same species of binding partner, singulated fromtheir particular batch, regrouped with like optical fibers from otherbatches having other species of binding partners. Upon regrouping of theoptical fibers, high density arrays are formed which can simultaneouslyinterrogate samples for a multiplicity of analytes for sampleidentification and processing.

BACKGROUND OF THE INVENTION

Biosensors are sensors that detect chemical species with highselectivity on the basis of molecular recognition rather than thephysical properties of analytes. See, e.g., Advances in Biosensors,A.P.F. Turner, Ed. JAI Press, London, (1991). Many types of biosensingdevices have been developed in recent years, including enzymeelectrodes, optical immunosensors, ligand-receptor amperometers, andevanescent-wave probes. Updike and Hicks, Nature, 214: 986 (1967),Abdel-Latif et al., Anal. Lett., 21: 943 (111988); Giaever, J. Immunol.,110: 1424 (1973); Sugao et al. Anal. Chem., 65: 363 (1993), Rogers etal. Anal. Biochem., 182: 353 (1989).

Biosensors comprising a biological "binding molecule" attached to anoptical fiber are well known in the prior art, most typically asevanescent wave detectors (see, for example, U.S. Pat. Nos. 4,447,546 toHirschfeld and 4,582,809 and 4,909,990 to Block et al.). In order tomaximize sensitivity and selectivity such biosensors typically utilize asingle species of biological binding molecule affixed to the face of thesensor.

Such "single-species" biosensors are limited in that they have noinherent means to correct or calibrate for non-specific binding. Thus,they must be calibrated against an external standard. In addition, theyare limited to the detection of a single analyte.

Biosensors comprising two or more species of biological binding partnersovercome these limitations. A "multi-species" biosensor in principlepermits simultaneous detection of as many different types of analytes asthere are species of biological binding partner incorporated into thesensor. In addition, comparison of the amounts of a single analytebinding to multiple species of binding partner provides a measure ofnon-specific binding and thus acts as an intrinsic control formeasurement variability introduced by non-specific binding.

In addition, the inclusion of fibers bearing biological binding partnersspecific for various analytes known to create a background signal in aparticular assay provides a means for simultaneously measuring andsubstracting out the background signal. The provision of a multiplicityof fibers bearing different species of binding partner allows thedetection of a multiplicity of moieties contributing to a background, orother, signal and the dissecting out of the contribution of each moietyto that signal.

To be most useful, a multi-species biosensor requires that the sensorprovide a separate signal characterizing binding of analytes to each ofthe various species of binding partner comprising the probe. Thus eachspecies of binding partner must be individually "addressed".

In addition, a "sensor face" (the surface bearing the biological bindingpartners) that has a relatively small surface area will facilitatemeasurement of small sample volumes as less sample material will berequired to fully immerse the sensor face. A small surface area detectorwill also prove advantageous for use in in vitro measurements.Preparation of a detector bearing a large number of different biologicalbinding partners that occupies a small area may be viewed as thepreparation of a high density array of biological binding partners.

The creation of high density arrays of biological binding partners whereeach species of binding partner is uniquely addressed presentsformidable fabrication problems. One of the most successful approaches,to date, is the large scale photolithographic solid phase synthesispioneered by Affymax Inc. (see, e.g., Fodor et al. Science 251: 767-773(1991) and U.S. Pat. No. 5,143,854). In this approach arrays of peptidesor nucleic acids are chemically synthesized on a solid support.Different molecules are simultaneously synthesized at differentpredetermined locations on the substrate by the use of aphotolithographic process that selectively removes photolabileprotecting groups on the growing molecules in particular selectedlocations of the substrate. The resulting array of molecules is"spatially addressed". In other words the identity of each biologicalmolecule is determined by its location on the substrate.

The photolithographic approach, however, is limited to molecules thatcan be chemically synthesized. Thus, it is typically restricted topeptides shorter than about 50 amino acids and nucleic acids shorterthan about 150 base pairs. In addition, the photolithographic approachtypically produces such arrays on a planar substrate (e.g. a glassslide) and provides no intrinsic mechanism by which a signal produced bythe binding of a particular biological binding partner may betransmitted.

U.S. Pat. No. 5,250,264 to Walt et al. discloses a sensor comprising afiber optic array using a "plurality of different dyes immobilized atindividual spatial positions on the surface of the sensor." Each dye iscapable of responding to a different analyte (e.g., pH, O₂, CO₂, etc.)and the sensor as a whole is capable of providing simultaneousmeasurements of multiple analytes.

Although the sensor disclosed by Walt et al. is not a biosensor, thereference describes a means of fabricating a sensor bearing a pluralityof uniquely addressed "detection moieties". In Walt et al. opticalfibers are first assembled to form a bundle. Transmission ends of afiber or group of fibers of are then specifically illuminated. Eachilluminated fiber transmits the light to its respective sensor end wherethe light "photopolymerizes" a sensor dye mixture causing the dye tobind to the sensor end. This process is repeated with different fibersfor different photopolymerized dyes. This repetition continues until asensor array is constructed.

This approach suffers from the limitations that it requiresphotopolymerizable sensor dyes and thus is limited in the number ofdifferent species per probe by the number of different dye type. Inaddition, this reference provides no means for attaching uniquelyaddressed biological molecules (e.g. peptides, nucleic acids,antibodies) to the sensor. Thus Walt et al. provide no means for thefabrication of biosensors.

SUMMARY OF THE INVENTION

The present invention provides a novel means for fabricating biosensorscomprising a plurality of biological "binding partners" (molecules thatspecifically bind other molecules to form a binding complex such asantibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid,biotin-avidin, etc.) linked to optical fibers. In particular the methodprovides a means of preparing a high density array of biological bindingpartners where each species of binding partner is uniquely addressed. Incontrast to certain methods in the prior art the biological bindingpartners utilized in the present invention are not limited to chemicallysynthesized oligonucleotides or peptides, but rather include nucleicacids, antibodies, proteins, lectins and other binding partners derivedfrom cells, tissues or organisms in their native state or otherwisemodified through the methods of recombinant DNA technology.

In particular, the biosensors of the present invention comprise amultiplicity of optical fibers bundled together to form an optical fiberarray. The sensor end of each optical fiber or group of optical fiberscomprising the optical fiber array bear a particular species ofbiological binding partner. Optical signals produced by binding of ananalyte to a biological binding partner are conducted along therespective optical fibers to a transmission end which may be attached toa detector. Detection of the signal from the fibers corresponding toeach species of biological binding partner provides a simultaneousmeasurement of the binding of a multiplicity of analytes.

The present invention provides a method of fabrication of fiber opticbiosensors. The method involves providing a multiplicity of opticalfibers which are grouped into a plurality of separate fiber groups orbatches. Each fiber has a sensor end and a transmission end and thefibers in each group are oriented so that the sensor ends are commonlyaligned. Each group of fibers is then treated to attach a single speciesof biological binding partner to the sensor ends of the constituentfibers. Alternatively, a multiplicity of species of biological bindingpartner may be attached to each group as long as the multiplicity ofspecies of biological binding partners attached to one fiber group isdifferent than the multiplicity of species attached to the other fibergroups.

Fibers or groups of fibers are then selected and discretely separatedfrom their respective batches. One or more of the discretely separatedfibers from each group are then recombined at their sensor ends withother fibers from other batches to produce an optical fiber array. Thesensor ends may be arranged in a substantially planar orientation or maybe tiered to form a tiered sensor face. The optical fiber array containsfibers capable of assaying simultaneously the binding of components of atest sample to the various binding partners on the different fibers ofoptical fiber array.

The batch identity of each fiber is maintained during the bundlingprocess, preferably at or adjacent to the transmission end of thefibers. These transmission ends are then discretely addressed todetectors--such as a multiplicity of optical sensors. The location andspatial array of the transmission ends corresponding to particularbiological binding partners are distinct from one another and known.

Thus, the invention provides for the fabrication of a high density arrayof biological binding partners in which each binding partner is uniquelyaddressed. An optical signal, produced by binding of the binding partnerto its substrate to form a binding complex, is conducted through theoptical fiber or group of fibers to a detector for each discrete test.Thus, binding of a molecule to a particular biological binding partneris specifically detectable. By examining the addressed transmission endsof fibers, or groups of fibers, the addressed transmission ends cantransmit unique patterns assisting in rapid sample identification ofanalytes by the sensor.

In one embodiment, the fiber optics might bear nucleic acid bindingpartners to which nucleic acids in the test sample might hybridize. Asused herein, the terms polymer in either single- or double-strandedform, and unless otherwise limited, would encompass known analogs ofnatural nucleotides that can function in a similar manner as naturallyoccurring nucleotides.

In one particularly preferred embodiment, the biosensor comprises aplurality of fibers, each fiber including an sensor end and atransmission end, the sensor end of at least one first fiber havingattached a first biological binding partner and the sensor end of atleast one second fiber having attached a second biological bindingpartner, a transmission array having first and second positionsaddressing the transmission ends of the first and second fibers, meansfor addressing the transmission ends of the first and second fibers tothe transmission array, optical interrogation means adjacent thetransmission ends for examining the comparative attachment of analytesat the sensor ends of the fibers. The sensor ends of the first andsecond fibers may be arranged to form a tiered sensor face. The firstand second binding partners may be nucleic acids, for example, DNA andcDNA, and the nucleic acids may be mapped to specific regions on one ormore human chromosomes. In a particularly preferred embodiment, thetarget nucleic acids are about 1,000 to 1,000,000 nucleotides incomplexity.

Nucleic acid bearing arrays are particularly useful in ComparativeGenomic Hybridization (CGH) assays to detect chromosomal abnormalities;in particular increases or decreases in copy number of particularchromosomal regions. In one example of this approach, a first collectionof (probe) nucleic acids is labeled with a first label, while a secondcollection of (probe) nucleic acids is labeled with a second label. Abiosensor, as described above, is one in which the biological bindingpartners are target nucleic acids. (As used herein the term "targetnucleic acids" typically refers to nucleic acids attached to the opticalfibers comprising the fiber optic array, while "probe nucleic acids" arethose nucleic acids free in solution that hybridize with the targetnucleic acids.) The ratio of hybridization of the nucleic acids isdetermined by the ratio of the two (first and second) labels binding toeach fiber in the array. Where there are chromosomal deletions ormultiplications, differences in the ratio of the signals from the twolabels will be detected. Identification of the specific optical fibersin the array giving rise to these ratios will indicate the nucleic acidsequence the probe bears and thus the nucleic acid sequence that isaltered.

The target nucleic acids (the nucleic acids attached to the opticalfibers) may include DNA and cDNA and may be mapped to specific regionsin human chromosomes. In addition, the target nucleic acids arepreferably about 1,000 to 1,000,000 nucleotides in complexity. Thecomplexity of the sequence complementary to the target nucleic acid ispreferably less than 1% of the total complexity of the sequences in thesample.

The first and second labels are preferably fluorescent labels. In aparticularly preferred embodiment, the first probe nucleic acidscomprise mRNA or cDNA from a test cell and the second probe nucleicacids comprise mRNA or cDNA from a reference cell. In another preferredembodiment, the first probe nucleic acids are from a test genome and thesecond probe nucleic acids are from a reference genome. The test genomemay comprise nucleic acids from fetal tissue or from a tumor.

According to one aspect of the invention, arrays of optical fibers aredisclosed where the interrogating end of each fiber in the arraycomprises a multiplicity of biological "binding partners." Each bindingpartner is attached to one or more optical fibers specifically addressedor identified at the transmitting end as being connected to theparticular binding partner. With the transmission ends properlyaddressed and interrogated, measurement occurs.

In one specific embodiment, arrays of optical fibers bearing nucleicacid molecules are disclosed. These optical fibers at theirinterrogating ends have specific nucleic acids such as nucleic acidshaving a certain minimum length (e.g. 400 bp), or being derived fromparticular libraries (e.g. evenly spaced along a particular chromosomeor representing a particular gene).

It is an advantage of the disclosed apparatus and process that theconstructed array can be tailored to rapid screening of extensive arraysof biological binding partners. Using already identified information,arrays can be assembled which can simultaneously and rapidly surveysamples nucleic acid variations across entire genomes. For example, afiber optic sensor bearing 30,000 target nucleic acids, each containing100 kb of genomic DNA could give complete coverage of the human genome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing a group of fibers with thesensor ends bound together for joint treatment to attach a bindingpartner and the transmission ends discretely marked to enable the fibersof the illustrated group to be distinguished from fibers of similargroups when subsequent separation of the fibers later occurs from thegroup.

FIG. 2 illustrates a plurality of differing batches of treatmentsolution with the sensor ends of the group of fibers of FIG. 1 beingimmersed for treatment in one of the fiber batches;

FIG. 3 illustrates differing groups of previously treated fibers lyingside-by-side with fibers being singulated from each group for gatheringinto a common high density array.

FIG. 4 illustrates an assembled high density array at the sensor andtransmitting ends only with disposition of the sensor ends in a tiereddisposition for transillumination by interrogating light and thedetector ends identified and discretely addressed to a sensor array, thesensor array here illustrated having a corresponding array of condensinglenses for relaying fiber illumination to a detector surface;

FIG. 5 illustrates a comparative genomic hybridization process beingcarried out with two samples for comparison having been previouslytagged with differing fluorophores and being added together in a commonbatch.

FIGS. 6A and 6B illustrate an expanded detail of the common batch ofFIG. 5 being transilluminated at the tiered sampling fibers at thesensor end of the array for the excitation of fluorophores attached tothe binding partners without undue direct illumination of the fibersthemselves.

FIGS. 7A and 7B illustrate a detector that may be used in conjunctionwith an optical fiber array or with any other array of light sources(e.g. an array of hybridized fluorescent probes).

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is a marked improvement in fiber optic biosensors,methods for fabricating biosensors, and methods for performingqualitative and quantitative measurements of biological molecules usinga unique fiber optic biosensor. In particular, the present inventionprovides for a novel method of construction of a biosensor comprising ahigh-density array of biological binding partners.

The biosensors of the present invention generally comprise a bundle ofcoalligned optical fibers. Each individual optical fiber or group offibers within the biosensor bears a single species of biological bindingpartner. As used herein biological binding partners are molecules thatspecifically recognize and bind other molecules thereby forming abinding complex. Typical binding complexes include, but are not limitedto, antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid,biotin-avidin, receptor--receptor ligand, etc.

The terms "specifically recognize and bind" refers to the binding of abiological binding partner to a particular molecule and to no othermolecule to which the biological binding partner is normally exposed. Inthe case of nucleic acids, specific binding is by hybridization and theterms "specific hybridization" or "specifically hybridizes with" refersto hybridization in which a probe nucleic acid binds substantially totarget nucleic acid and does not bind substantially to other nucleicacids present in the biosensor under defined stringency conditions. Oneof skill will recognize that relaxing the stringency of the hybridizingconditions will allow sequence mismatches to be tolerated. The degree ofmismatch tolerated can be controlled by suitable adjustment of thehybridization conditions.

The term "species", as used herein, refers to a biological bindingpartner capable of specifically binding a particular target molecule.Thus, for example, biological binding partners may both be nucleicacids, but if they have different nucleotide sequences, so that theyspecifically hybridize to different molecules, they are considereddifferent species. Similarly, two antibodies specific for differentepitopes are considered different species.

In a preferred embodiment, the biosensor bears two or more differentspecies of biological binding partner. The use of two or more species ofbinding partner permits the simultaneous detection of two or moreanalytes in a test sample with the number of detectable analytes limitedonly by the number of different biological binding partners present onthe biosensor. The biosensor may optionally include additional opticalfibers lacking biological binding partners. These additional fibers maybear moieties for the detection of various physical parameters of thetest sample, such as temperature or pH, or alternatively may lack anymoiety and simply serve as an optical conduit for visualization therebyserving as an endoscope for guiding the insertion of the biosensor probein various in vivo applications.

A biosensor bearing a plurality biological binding partners permits thesimultaneous assay of a multiplicity of analytes in a sample. Inaddition, the measurement of binding of a single analyte to a number ofdifferent species of biological binding partners provides a control fornon-specific binding. A comparison of the degree of binding of differentanalytes in a test sample permits evaluation of the relative increase ordecrease of the different analytes. Finally, because of the smallcross-sectional area of optical fibers, the bundling together into anoptical array a number of optical fibers, each bearing a differentbiological binding partner, provides an effective mechanism for thefabrication of high density arrays of biological binding partners forsuitable for a wide variety of in vivo and in vitro assays.

I. Organization of the Biosensor.

The unique fiber optic biosensor of the present invention, itsorganization, its construction and its component parts are illustratedby FIGS. 1-6 respectively. Each discrete fiber optic biosensor iscomprised of a plurality of fiber optic strands 10 disposed coaxiallyalong their lengths to form a single, discrete construction. Thebiosensor thus comprises an optical fiber array 14, the smallest commonrepeating unit within which is a single fiber optical strand 10.

A preferred fiber optical biosensor is illustrated by FIG. 4. As seentherein an individual fiber optical strand 10 comprises a single opticalfiber having a rod-like shaft and two fiber ends designated a sensor end11 and a transmission end 12 each of which provides a substantiallyplanar end surface. The optical fiber strand 10 is typically composed ofglass or plastic and is a flexible rod able to convey light energyintroduced at either of its ends 11, 12. Such optical fibers 10 areconventionally known and commercially available. Alternatively, the usermay himself prepare optical fibers in accordance with the conventionalpractices and techniques reported by the scientific and industrialliterature. Accordingly, the optical fiber 10 is deemed to beconventionally known and available as such.

It will be appreciated that FIGS. 1-6 are illustrations in which thefeatures have been purposely magnified and exaggerated beyond theirnormal scale in order to provide both clarity and visualization ofextreme detail. Typically, the conventional optical fiber has a crosssection diameter of 5-500 micrometers and is routinely employed inlengths ranging between centimeters (e.g. in the laboratory) tokilometers (e.g. in field telecommunications). Typically, however, whenutilized in a biosensor, the optical fibers will preferably range inlength from centimeters to about a meter.

Although the optical fiber 10 is illustrated via FIGS. 1-4 as acylindrical extended rod having substantially circular end surfaces,there is no requirement or demand that this specific configuration bemaintained. To the contrary, the optical fiber may be polygonal orasymmetrically shaped along its length, provide special patterns andshapes at the sensor end or transmission end and need not present an endsurface that is substantially planar. Nevertheless, in a preferredembodiment, the optical fiber is substantially cylindrical.

Each optical fiber 10 may be individually clad axially along its length.The cladding may be any material which has a lower refractive index andprevents the transmission of light energy photons from the optical fiber10 to the external environment. The cladding may thus be composed of avariety of different chemical formulations including various glasses,silicones, plastics, cloths, platings and shielding matter of diversechemical composition and formulation. Methods of cladding includingdeposition, extrusion, painting and covering are scientifically andindustrially available and any of these known processes may be chosen tomeet the requirements and convenience of the user.

The user has a variety of choices at his discretion regarding theconfiguration of the sensor end 11 of the optical fiber 10. As indicatedabove, the sensor end 11 may present a surface that is substantiallyplanar and smooth. Alternatively the sensor end 11 may provide an endsurface which is essentially convex or concave.

It will be appreciated that the range and variety of dimensional andconfigurational variation of the optical fiber 10 is limited only by theuser's ability to subsequently dispose and immobilize a biologicalbinding partner on the intended sensor end 11 of the strand. The use ofconcave or convex sensor ends 11 will provide greater surface area uponwhich to immobilize a biological binding partner thereby increasingefficiency (the signal to noise ratio per optical fiber) of thebiosensor.

While the single repeating component of the fiber optic biosensor is theindividual optical fiber 10, it is the aggregation of a plurality ofsuch fibers to form a discrete optical fiber array 14 that permits thesimultaneous detection of a multiplicity of analytes. When the opticalfibers are aggregated to form a discrete optical fiber array 14, thecoalligned sensor ends 11 of the fibers are aggregated to form a sensorface 13. A typical biosensor is illustrated in FIG. 4 and FIGS. 6A and6B in which the sensor face 13 appears in exaggerated, highly simplifiedviews without regard to scale. The optical fiber array 14 comprises aunitary rod-like collective body forming a sensor face 13 and a and atransmission face 15.

In practice, it is estimated that there are typically 1000-3000 fiberoptical strands in a conventional imaging fiber of 0.5 mm diameter andnearly 1 million strands per square millimeter. The total number ofindividual fiber optic strands forming the optical fiber array 14 of thepresent invention will be approximately as great; the total numbervarying with the cross-sectional diameter of each optical fiber, thepattern of packing of the individual optical fibers in the collectivebody, and the thickness of cladding material, when employed. It will beappreciated that a 1 square millimeter biosensor, containing nearly 1million strands in which groups of about 33 optical fibers are eachlabeled with a different species of biological binding partner willproduce a sensor face 13 comprising approximately 30,000 differentspecies of biological binding partner in 1 square millimeter. Asexplained above, such a sensor could provide nucleic acid biologicalbinding partners covering the entire human genome at 10 megabaseintervals.

The sensor face 13 need not be arranged as a planar surface. Rather, theindividual optical fibers may be "tiered" so as to protrude from theoptical array varying distances. This will maximize the exposure of eachoptical fiber sensor end 11 both to the sample fluid and to atransilluminating light source 19 as shown in FIGS. 6A and 6B.

In a preferred embodiment, the sensor ends 11 of the optical fiberscomprising the optical fiber array 14 will be bundled together in arandom or haphazard pattern to form the sensor face 13. Alternatively,the placement of the sensor ends 11 may be highly ordered with thesensor end of each fiber occupying a specific predetermined location inthe sensor face 13. As indicated above, the sensor ends 11 of theoptical fibers 10 forming the sensor face 13 or the optical fiber array14 have attached a biological binding partner.

Each optical fiber or group of fibers comprising the optical fiber array14 may bear a different species of biological binding molecule. Althoughthe use of a single species of biological binding partner per opticalfiber or group of fibers is preferred, alternatively, each optical fiberor group of fibers may bear a multiplicity of biological bindingpartners as long as that multiplicity differs from the biologicalbinding partners or multiplicity of biological binding partners presenton other fibers or groups of fibers comprising the optical fiber array14. The fibers bearing like species of binding partner may be physicallygrouped together thereby producing distinct regions of the sensor face13 characterized by the presence of a particular biological bindingpartner or alternatively the fibers bearing different binding partnersmay be intermingled, the sensor face 13 presenting a relatively uniformor haphazard or random distribution of species of biological bindingpartners.

The transmission face 15 of the optical array may present asubstantially planar optical array lacking any further attachments.However, in a preferred embodiment, the transmission face 15 will bepermanently or removeably attached to a detector 20, as illustrated inFIG. 4. The detector may comprise one or more lenses for focusing andenhancement of an optical signal transmitted along the optical fiberscomprising the optical fiber array 14. The detector may additionallycomprise a device for transforming the optical signal into a digital oranalog electrical signal. Preferred detectors include phototubes(photomultipliers) or charge coupled devices (CCDs). A singlephotomultiplier or CCD element may be arranged to measure the aggregatesignal provided by the entire transmission face 15 of the biosensor.Alternatively, a CCD (or other) camera may be focused at thetransmission face of the biosensor to simultaneously read signals fromall of the optical fibers while permitting individual evaluation of thesignal from each fiber or group of fibers. In another embodiment,multiple CCD elements or phototubes are used to each detect a signalrepresenting binding of a single species of biological binding partnerpresent at the sensor face 13 of the biosensor. Thus the detector ispreferably arranged to read the signal from single optical fibers 10 orfrom groups of optical fibers where all of the optical fibers 10 in agroup bear the same species of biological binding partner.

In addition to detecting optical signals from a fiber optic array, thedetector 20, may be generally used to amplify and detect optical signalsfrom any array of light sources. Thus, for example, the array of lightsources may be an array of fluorescent spots as due to hybridization offluorescently labeled probes hybridized to arrays of target nucleicacids. Similarly, the array may be of fluorescently labeled antibodiesbound to an array of proteins to which the antibodies bind, orconversely fluorescently labeled proteins bound to an array ofantibodies.

In a preferred embodiment suitable for such applications, illustrated inFIGS. 7A and 7B, the detector 20 may comprise a compound objective lens31 that consists of an array of single lenses 32. The single lenses arespaced so that each lens is focused on a location 34 in the array wherefluorescence is to be measured. The detector may optionally include abeam splitter 35 a second lens 36 an optical filter 37 and a detectiondevice such as a camera 38. The beam splitter is then used to direct anexcitation illumination 39 upon the array of light sources. Theresulting fluorescence at each spot is then focused through the compoundobjective lens 31 optionally focused by a second lens, optionallyfiltered by an optical filter and then detected either visually or by adetection means such as a camera.

The compound objective may be cast, pressed, etched, or ground out ofglass, plastic, quartz, or other materials well known as suitable forlens manufacture. The compound lens may be formed as a single piece, oralternatively may be assembled by attaching together simple lenses toform a compound objective.

II. Fabrication of the Biosensor

FIGS. 1-4 illustrate a method of fabrication of a biosensor comprising aplurality of optical fibers bearing biological binding partners. Ingeneral the method involves providing a plurality of optical fibers witheach fiber having a sensor end and a transmission end with a particularspecies of biological binding partner attached to the sensor end of eachfiber. Fibers with differing binding partners are combined to form anoptical fiber array wherein said fibers have commonly aligned sensorends for simultaneous assay of a sample. The transmission ends of thecombined discrete fibers are addressed for interrogation to produce thefiber optic sensor.

FIG. 1, illustrates a particularly preferred embodiment that details onemethod of providing the optical fibers with attached biological bindingpartners. A plurality of optical fibers 10 are provided, each fiberhaving a sensor end 11 and a transmission end 12. The fibers arearranged together to form a plurality of fiber groups or bundles 16, asshown in FIG. 2, with the fibers in each bundle disposed coaxiallyalongside each other with the sensor ends 11 of each fiber commonlyaligned at the same end of the bundle. The fibers comprising each bundlemay be optionally marked 17 to permit their identification whensubsequently removed from the bundle.

As shown in FIG. 2, each bundle of fibers is separately treated toattach a particular species of biological binding partner 18 to thesensor ends 11 of the optical fibers comprising the particular bundle.Many methods for immobilizing biological binding partners 18 on avariety of solid surfaces are known in the art. In general, the desiredcomponent may be covalently bound or noncovalently attached throughnonspecific binding.

In preparing the sensor end 11 for attachment of the binding partner, aplurality of different materials may be employed, particularly aslaminates, to obtain various properties. For example, proteins (e.g.,bovine serum albumin) or mixtures of macromolecules (e.g., Denhardt'ssolution) can be employed to avoid non-specific binding, simplifycovalent conjugation, enhance signal detection or the like.

If covalent bonding between a biological binding partner and the surfaceof the sensor end 11 is desired, the surface will usually bepolyfunctional or be capable of being polyfunctionalized. Functionalgroups which may be present on the surface and used for linking caninclude carboxylic acids, aldehydes, amino groups, cyano groups,ethylenic groups, hydroxyl groups, mercapto groups and the like.

Covalent linkage of the binding partner to the sensor end may be director through a covalent linker. Generally linkers are either hetero- orhomo-bifunctional molecules that contain two or more reactive sites thatmay each form a covalent bond with the respective binding partner.Linkers suitable for joining biological binding partners are well knownto those of skill in the art. For example, biological binding partnersmay be joined by a peptide linker, by a straight or branched chaincarbon chain linker, or by a heterocyclic carbon. Heterobifunctionalcross linking reagents such as active esters of N-ethylmaleimide havebeen widely used. See, for example, Lerner et al. Proc. Nat. Acad. Sci.(USA), 78: 3403-3407 (1981) and Kitagawa et al. J. Biochem., 79: 233-236(1976), which are incorporated herein by reference.

The manner of linking a wide variety of compounds to various surfaces iswell known and is amply illustrated in the literature. Proteins, forexample, may be joined to linkers or to functional groups on the sensorend 11 by coupling through their amino or carboxyl termini, or throughside groups of various constituent amino acids. Thus, coupling through adisulfide linkage to a cysteine is common.

Similarly, methods for immobilizing nucleic acids by introduction ofvarious functional groups to the molecules is known (see, e.g., Bischoffet al., Anal. Biochem. 164:336-344 (1987); Kremsky et al., Nuc. AcidsRes. 15:2891-2910 (1987) which are incorporated herein by reference).Modified nucleotides can be placed on the target using PCR primerscontaining the modified nucleotide, or by enzymatic end labeling withmodified nucleotides.

Referring to FIG. 3, after the biological binding partners 18 areattached to the sensor faces 11 of the optical fibers 10 comprising eachbundle, individual fibers, or groups of fibers, are separated from eachbundle. In FIG. 3, only four fiber bundles F₁ -F₄ are illustrated. Inthe process of being separated from each of the respective fiber bundlesF₁ -F₄ are individual fibers 10_(a) -10_(d). These respective fibers arebeing regrouped into optical fiber array 14.

The individual fibers or groups of fibers, may be marked prior toseparation from the original bundle to facilitate identification of thebinding partner bound to a particular fiber or group during laterassembly steps. The separated fibers, or groups of fibers, arerecombined with fibers or groups of fibers, separated from differentbundles to form an optical fiber array 14 comprising a plurality offibers or groups of fibers where each fiber or group of fibers bears adifferent species of biological binding partner.

FIG. 4 illustrates that members of the optical fiber array 14 areoriented such that the sensor ends 11 of all of the constituent opticalfibers 10 are commonly aligned at the same end of the optical fiberarray 14 thereby forming a sensor face 13. The fibers may be arranged ina substantially planar configuration or tiered, as illustrated in FIGS.6A and 6B.

The fibers may be bundled at the sensor face 13 in a substantiallyrandom or haphazard manner with the relative location of a the sensorend 11 of a particular fiber within the sensor face 13 being determinedby chance. Alternatively, the fibers may be positioned within the fiberarray in a highly ordered manner such that the location of anyparticular optical fiber 10 in the sensor face 13 is predetermined.

The transmission ends 12 of the optical fibers comprising the opticalfiber array 14 are addressed to permit interrogation and detection ofbinding events to the biological binding partners attached to the sensorface 13. Addressing is accomplished by any of a number of means wellknown to those of skill in the art. In a preferred embodiment, thetransmission ends 12 of individual optical fibers, or groups of opticalfibers all bearing the same species of biological binding partner, arespatially addressed. This comprises localizing the optical fibers orbundles of optical fibers at fixed locations relative to the otheroptical fibers or bundles of optical fibers comprising the optical fiberarray 14, see e.g. FIG. 4. Most typically this may be accomplished byattaching the fiber array to a fiber optic connector and ferrule (e.g.see AMP, Inc. Harrisburg, Pa.).

Alternatively, the transmission ends 12, may be addressed by attachingthe transmission end of each optical fiber 10 or bundle of opticalfibers bearing a particular biological binding partner to an individualdetector. Each detector is subsequently known to be associated with aparticular biological binding partner and there is no need to preserve afixed spatial relationship between any of the transmission ends 12.

Detection of a signal from the biosensor (optical array) may beaccomplished by visual inspection of the transmission face 15 of theoptical fiber array 14 or by the use of one or more detectors 20. Asindicated above, the transmission face may be permanently or removablyattached to a single optical lens or system of multiple optical lenses.The lens or lenses may be arranged to focus an optical signal from theentire transmission face 15 or from selected subregions of thetransmission face. In a preferred embodiment, lenses will be arranged toeach focus an optical signal from the portion of the transmission face15 corresponding to a single biological binding partner. In the extremecase, the signal for each optical fiber comprising the optical fiberarray 14 will be individually focused.

Again with a lens or lens system present, the signal may be simplydetected visually. However, in a preferred embodiment, the use ofdetectors is contemplated. Preferred detectors are devices that convertan optical signal into a digital or analog electrical signal. Typicallydetectors are of two general types: phototubes and charge coupleddevices (CCDs). A single photomultiplier or CCD element may be utilizedto measure the aggregate signal provided by the entire transmission face15 of the biosensor. More preferably, however, multiple CCD elements orphototubes are used to each detect a signal representing binding of asingle species of biological binding partner present at the sensor face13 of the optical fiber array 14.

The detector system may be employed with a computerized data acquisitionsystem and analytical program. In this embodiment, providing a fullyautomated, computer controlled processing apparatus and measurementsystem, the data obtained from the biosensor is processed intoimmediately useful information. By using such fully automated,computerized apparatus and analytical systems, not only are a variety ofdifferent measurements made and diverse parameters measured concurrentlywithin a single fluid sample, but also many different fluid samples maybe analyzed individually seriatim for detection of multiple analytes ofinterest concurrently--each individual fluid sample following itspredecessor in series.

II. Methods of Use

A variety of in vitro measurements and analytical determinations may bemade using a fiber optic biosensor prepared in accordance with thepresent invention. In vitro applications and assay techniques may beperformed concurrently using one or multiple fluid samples. Eachconcurrently conducted measurement or determination for differentanalytes of interest is made individually, accurately and precisely. Theobserved results are then correlated and/or computed to provide preciseinformation regarding a variety of different parameters or ligandsindividually.

The fiber optic biosensor of the present invention may also be employedin a variety of different in vivo conditions with both humans andanimals. The present invention provides accurate and precisemeasurements and determinations using a single discrete fiber opticbiosensor rather than the conventional bundle of different sensorsjoined together for limited purposes. The present invention thusprovides a minimum-sized diameter sensor for in vivo catheterization: aminimum intrusion into the bloodstream or tissues of the living subjectfor assay purposes, and a minimum of discomfort and pain to the livingsubject coupled with a maximum of accuracy and precision as well as amultiplicity of parameter measurement in both qualitative and/orquantitative terms.

The biosensor of the present invention may be utilized for the detectionof a wide variety of analytes, depending on the particular biologicalbinding partner selected. As indicated above, biological bindingpartners are molecules that specifically recognize and bind othermolecules thereby forming a binding complex. Typical binding complexesinclude, but are not limited to, antibody-antigen, lectin-carbohydrate,nucleic acid-nucleic acid, biotin-avidin, receptor-receptor ligand, etc.Either member of the binding complex may be used as the biologicalbinding member attached to the sensor end 11 of the optical fiberscomprising the biosensor. Thus, for example, where it is desired todetect an antibody in a sample, the corresponding antigen may beattached to the sensor end. Conversely, where it is desired to detectthe antigen in the sample, the antibody may be attached to the sensorend.

The selection of binding partners for a particular assay is well knownto those of skill in the art. Typically, where proteins are to bedetected, antibodies are most preferred as the biological bindingpartner. Where enzymatic substrates are to be detected, enzymes arepreferred biological binding partners, and where nucleic acids are to bedetected, nucleic acid binding partners are most preferred. Thus, forexample, fiber optic biosensors have been described that utilize enzymessuch as xanthine oxidase and peroxidase to detect hypoxanthine andxanthine (Hlavay et al., Biosensors and Bioelectronics, 9(3): 189-195(1994), that use alkaline phosphatase to detect organophosphorous-basedpesticides (Gao et al. Proceedings--Lasers and Electro-Optics Society,Annula Meeting, 8(4): abstract 20782 (1994), and that use antibodies orDNA binding proteins (Anderson, et al., Fiber Optic Medical andFluorescent Sensors and Applications, Proc. S.P.I.E. 1648: 39-43 (1992).

Of course the biosensor may be designed to simultaneously detect severaldifferent classes of analyte. Thus the sensor may bear a combination oftwo or more different classes of biological binding partner. The sensorface 13 will bear one or more binding partners selected, for example,from the group consisting of nucleic acids, proteins, antibodies,carbohydrates, biotin, avidin, and lectins.

In the simplest application, the biosensor of the present invention maybe utilized to detect a single analyte in a test sample. The test samplemay be in vivo, in culture, or in vitro. The assay may register simplepresence or absence of the analyte or may quantify the amount of analytepresent in the sample.

The assay may be run in either a direct or a competitive format. In adirect format, the amount of analyte is determined directly measuringthe analyte bound to the biological binding partner. In a competitiveformat, a known analyte is present in the sample and the test analyte isdetected by its ability to compete the known analyte from the biologicalbinding partners present on the sensor face 13.

In a preferred method of use, the optical fibers 10 comprising theoptical fiber array 14 conduct an optical signal indicative of thebinding between a biological binding partner on the sensor face 11 andthe analyte in the sample. The optical signal may be produced by anumber of means known to those of skill in the art. Typically theoptical signal is generated by a fluorescent, luminescent, orcolorimetric label present at the sensor end 11 of the optical fiber 10.Typically the concentration of label at the sensor end of the opticalfiber is a function of the concentration of analyte that specificallybinds to the biological binding partner present on that sensor end.

Methods of providing a label whose concentration is a function of theamount of an analyte specifically associated with a biological bindingpartner are well known to those of skill in the art. In the simplestapproach, the analyte itself is labeled. Binding of the analyte to thebinding partner then brings the label into proximity with the sensor end11 to which the binding partner is attached. Alternatively, a labeled"blocking" analyte may be provided in the test sample or pre-bound tothe biosensor. Displacement of the labeled "blocking" analyte by theunlabeled test analyte in the sample produces a reduction of label boundto the sensor end where the reduction is proportional to theconcentration of unlabeled analyte in the test sample.

Other approaches may use a second biological binding partner that itselfis labeled. The first biological binding partner attached to the sensorend binds and thereby immobilizes the analyte. The second, labelledbinding partner then binds to the analyte immobilized on the sensor endthereby bringing the label in close proximity to the sensor end where itmay be detected.

Luminescent labels are detected by measuring the light produced by thelabel and conducted along the optical fiber. Luminescent labelstypically require no external illumination.

In contrast calorimetric or fluorescent labels typically require a lightsource. Colorimetric labels typically produce an increase in opticalabsorbance and/or a change in the absorption spectrum of the solution.Colorimetric labels are measured by comparing the change in absorptionspectrum or total absorbance of light produced by a fixed light source.In the present invention, the change in light absorbance or absorptionspectrum is preferably detected through the optical fibers comprisingthe biosensor. The change in absorbance, or absorption spectrum, may bemeasured as a change in illumination from an absolutely calibrated lightsource, or alteratively may be made relative to a second "referencelight source". The light source may be external to the biosensor or maybe provided as an integral component. In one embodiment, some of theconstituent optical fibers will conduct light from the signal and/orreference source to the sensor face. For maximum sensitivity the lightused to measure absorbance, or absorption spectrum, changes will bedirected directly at the sensor face of the biosensor.

Fluorescent labels produce light in response to excitation by a lightsource. The emitted light, characteristically of a different (lower)wavelength than the excitation illumination, is detected through theoptical fiber to which the fluorescent label has become bound.

The excitation illumination may be provided by an integral component ofthe biosensor or by a separate light source according to a number ofmethods well known to those of skill in the art. Evanescent wave systemsinvolve introducing a light beam at the transmission end 12 of theoptical fiber. This light beam is conducted along the fiber until itreaches the sensor end 11 of the fiber where it generates in the testsolution an electromagnetic waveform known as the evanescent wavecomponent. The evanescent wave component may be sufficient to excite afluorophore and produce a fluorescent signal. (See, for example U.S.Pat. Nos. 4,447,546 and 4,909,990 which are incorporated herein byreference).

In another embodiment, the excitation illumination is provided externalto the biosensor. It is particularly preferred that the illumination beprovided as a "transillumination" normal to the sensor ends 11 of theoptical fibers (see, e.g. FIG. 4). This provides an increased signal tonoise ratio as, in this configuration, most of the excitationillumination will not be conducted along the optical fibers. Theindividual optical fibers 10 comprising the biosensor may be tiered, forexample, as shown in FIGS. 6A and 6B, to prevent individual fibers fromshadowing each other when transilluminated.

To optimize a given assay format one of skill can determine sensitivityof fluorescence detection for different combinations of optical fiber,sensor face configuration, fluorochrome, excitation and emission bandsand the like. The sensitivity for detection of analyte by variousoptical fiber array configurations can be readily determined by, forexample, using the biosensor to measure a dilution series offluorescently labeled analytes. The sensitivity, linearity, and dynamicrange achievable from the various combinations of fluorochrome andbiosensor can thus be determined. Serial dilutions of pairs offluorochromes in known relative proportions can also be analyzed todetermine the accuracy with which fluorescence ratio measurementsreflect actual fluorochrome ratios over the dynamic range permitted bythe detectors and biosensor.

Use in Comparative Genomic Hybridization

In a particularly preferred embodiment, the biosensor of the presentinvention will be used in a Comparative Genomic Hybridization (CGH)assay. Comparative genomic hybridization (CGH) is a recent approach usedto detect the presence and identify the chromosomal location ofamplified or deleted nucleotide sequences. (See, Kallioniemi et al.,Science 258: 818-821 (1992), WO 93/18186, and copending application U.S.Ser. No. 08/353,018, filed on Dec. 9, 1994, which are incorporatedherein by reference).

In the traditional implementation of CGH, genomic DNA is isolated fromnormal reference cells, as well as from test cells (e.g., tumor cells).The two nucleic acids (DNA) are labelled with different labels and thenhybridized in situ to metaphase chromosomes of a reference cell. Therepetitive sequences in both the reference and test DNAs may be removedor their hybridization capacity may be reduced by some means such as anunlabeled blocking nucleic acid (e.g. Cot-1). Chromosomal regions in thetest cells which are at increased or decreased copy number can bequickly identified by detecting regions where the ratio of signal fromthe two DNAs is altered. For example, those regions that have beendecreased in copy number in the test cells will show relatively lowersignal from the test DNA than the reference compared to other regions ofthe genome. Regions that have been increased in copy number in the testcells will show relatively higher signal from the test DNA.

In one embodiment, the present invention provides a CGH assay in whichthe biosensor of the present invention replaces the metaphase chromosomeused as the hybridization target in traditional CGH. Instead, thebiological binding partners present on the biosensor are nucleic acidsequences selected from different regions of the genome. The biosensoritself becomes a sort of "glass chromosome" where hybridization of anucleic acid to a particular binding partner is informationallyequivalent to hybridization of that nucleic acid to the region on ametaphase chromosome from which the biological binding partner isderived. In addition, nucleic acid binding partners not normallycontained in the genome, for example virus nucleic acids, can beemployed.

More particularly, in a CGH assay, the biosensor may be utilized inmethods for quantitatively comparing copy numbers of at least twonucleic acid sequences in a first collection of nucleic acid moleculesrelative to the copy numbers of those same sequences in a secondcollection, as illustrated in FIG. 5. The method comprises labeling thenucleic acid molecules in the first collection 25 and the nucleic acidmolecules in the second collection 26 with first and second labels,respectively thereby forming at least two collections of nucleic acidprobes. The first and second labels should be distinguishable from eachother.

As used herein, the term "probe" is thus defined as a collection ofnucleic acid molecules (either RNA or DNA) capable of binding to atarget nucleic acid of complementary sequence through one or more typesof chemical bonds, usually through hydrogen bond formation. The probesare preferably directly or indirectly labelled as described below. Theyare typically of high complexity, for instance, being prepared fromtotal genomic DNA or mRNA isolated from a cell or cell population.

The probes 30 thus formed are contacted, either simultaneously orserially, to a plurality of target nucleic acids, present on the sensorface 13 of the biosensor of array 14 under conditions such that nucleicacid hybridization to the target nucleic acids can occur. Here atranilluminating light source 19 is utilized. After contacting theprobes to the target nucleic acids the amount of binding of each probe,and ratio of the binding of the probes is determined for each species oftarget nucleic acid. Typically the greater the ratio of the binding to atarget nucleic acid, the greater the copy number ratio of sequences inthe two probes that bind to nucleic acid. Thus comparison of the ratiosof bound labels among target nucleic acid sequences permits comparisonof copy number ratios of different sequences in the probes.

In a preferred embodiment, the sequence complexity of each targetnucleic acid in the biosensor is much less than the sequence complexityof the first and second collections of labeled nucleic acids. The term"complexity" is used here according to standard meaning of this term asestablished by Britten et al. Methods of Enzymol. 29:363 (1974). See,also Cantor and Schimmel Biophysical Chemistry: Part III at 1228-1230for further explanation of nucleic acid complexity.

The methods are typically carried out using techniques suitable forfluorescence in situ hybridization. Thus, the first and second labelsare usually fluorescent labels.

To inhibit hybridization of repetitive sequences in the probes to thetarget nucleic acids, unlabeled blocking nucleic acids (e.g., Cot-1 DNA)can be mixed with the probes. Thus, the invention focuses on theanalysis of the non-repetitive sequences in a genome. However, use ofrepetitive sequences as targets on the biosensor and omiting theblocking nucleic acids would permit relative copy number determinationsto be made for repetitive sequences.

In a typical embodiment, one collection of probe nucleic acids isprepared from a test cell, cell population, or tissue under study; andthe second collection of probe nucleic acids is prepared from areference cell, cell population, or tissue. Reference cells can benormal non-diseased cells, or they can be from a sample of diseasedtissue that serves as a standard for other aspects of the disease. Forexample, if the reference probe is genomic DNA isolated from normalcells, then the copy number of each sequence in that probe relative tothe others is known (e.g., two copies of each autosomal sequence, andone or two copies of each sex chromosomal sequence depending on gender).Comparison of this to a test probe permits detection of variations fromnormal. Alternatively the reference probe may be prepared from genomicDNA from a primary tumor which may contain substantial variations incopy number among its different sequences, and the test probe mayprepared from genomic DNA of metastatic cells from that tumor, so thatthe comparison shows the differences between the primary tumor and itsmetastasis. Further, both probes may be prepared from normal cells. Forexample comparison of mRNA populations between normal cells of differenttissues permits detection of differential gene expression that is acritical feature of tissue differentiation. Thus in general the termstest and reference are used for convenience to distinguish the twoprobes, but they do not imply other characteristics of the nucleic acidsthey contain.

Target nucleic acids

The target nucleic acids comprising the biological binding partnersattached to the sensor ends 11 of the optical fibers 10 and the probesmay be, for example, RNA, DNA, or cDNA. The nucleic acids may be derivedfrom any organism. Usually the nucleic acid in the target sequences andthe probes are from the same species.

The "target nucleic acids" comprising biological binding partnerstypically have their origin in a defined region of the genome (forexample a clone or several contiguous clones from a genomic library), orcorrespond to a functional genetic unit, which may or may not becomplete (for example a full or partial cDNA). The target nucleic acidscan also comprise inter-Alu or Degenerate Oligonucleotide Primer PCRproducts derived from such clones. If gene expression is being analyzed,a target element can comprise a full or partial cDNA.

The target nucleic acids may, for example, contain specific genes or, befrom a chromosomal region suspected of being present at increased ordecreased copy number in cells of interest, e.g., tumor cells. Thetarget nucleic acid may also be an mRNA, or cDNA derived from such mRNA,suspected of being transcribed at abnormal levels.

Alternatively, target nucleic acids may comprise nucleic acids ofunknown significance or location. The array of such target nucleic acidscomprising the sensor face 13 of a biosensor of the present inventioncould represent nucleic acids derived from locations that sample, eithercontinuously or at discrete points, any desired portion of a genome,including, but not limited to, an entire genome, a single chromosome, ora portion of a chromosome. The number of target elements and thecomplexity of the nucleic acids in each would determine the density ofsampling. For example an biosensor bearing 300 different species oftarget nucleic acid (biological binding partners), each target nucleicacid being DNA from a different genomic clone, could sample the entirehuman genome at 10 megabase intervals. An array of 30,000 elements, eachcontaining 100 kb of genomic DNA could give complete coverage of thehuman genome.

Similarly, an array of target nucleic acids comprising nucleic acidsfrom anonymous cDNA clones would permit identification of those thatmight be differentially expressed in some cells of interest, therebyfocusing attention on study of these genes.

In some embodiments, previously mapped clones from a particularchromosomal region of interest are used as targets. Such clones arebecoming available as a result of rapid progress of the worldwideinitiative in genomics.

Mapped clones can be prepared from libraries constructed from singlechromosomes, multiple chromosomes, or from a segment of a chromosome.Standard techniques are used to clone suitably sized fragments invectors such as cosmids, yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs) and P1 phage.

While it is possible to generate clone libraries, as described above,libraries spanning entire chromosomes are also available commercially.For instance, chromosome-specific libraries from the human and othergenomes are available from Clonetech (South San Francisco, Calif.) orfrom The American Type Culture Collection (see, ATCC/NIH Repository ofCatalogue of Human and Mouse DNA Probes and Libraries, 7th ed. 1993).

If necessary, clones described above may be genetically or physicallymapped. For instance, FISH and digital image analysis can be used toidentify and map the locations on a chromosome to which specific cosmidinserts hybridize. This method is described, for instance, in Lichter etal., Science, 247:64-69 (1990). The physically mapped clones can then beused to more finally map a region of interest identified using CGH orother methods.

One of skill will recognize that each target nucleic acids may beselected so that a number of nucleic acids of different length andsequence represent a particular region on a chromosome. Thus, forexample, a the sensor face 13 of the biosensor may bear more than onecopy of a cloned piece of DNA, and each copy may be broken intofragments of different lengths. One of skill can adjust the length andcomplexity of the target sequences to provide optimum hybridization andsignal production for a given hybridization procedure, and to providethe required resolution among different genes or genomic locations.Typically, the target sequences will have a complexity between about 1kb and about 1 Mb.

Preparation of probe nucleic acids

As with target nucleic acids (those attached to the fiber optic sensor),a wide variety of nucleic acids can be used as probe nucleic acids inthe methods of the present invention. The probes may be comprise, forexample, genomic DNA representing the entire genome from a particularorganism, tissue or cell type or may comprise a portion of the genome,such as a single chromosome.

To compare expression levels of a particular gene or genes, the probenucleic acids can be derived from mRNA or cDNA prepared from anorganism, tissue, or cell of interest. For instance, test cDNA or mRNA,along with mRNA or cDNA from normal reference cells, can be hybridizedto an array of target nucleic acids on the sensor comprising clones froma normalized cDNA library. In addition, probes made from genomic DNAfrom two cell populations can be hybridized to a target cDNA array todetect those cDNAs that come from regions of variant DNA copy number inthe genome.

The methods of the invention are suitable for comparing copy number ofparticular sequences in any combination of two or more populations ofnucleic acids. One of skill will recognize that the particularpopulations of sample nucleic acids being compared is not critical tothe invention. For instance, genomic or cDNA can be compared from tworelated species. Alternatively, levels of expression of particular genesin two or more tissue or cell types can be compared. As noted above, themethods are particularly useful in the diagnosis of disease.

Standard procedures can be used to isolate nucleic acids (either DNA ormRNA) from appropriate tissues (see, e.g., Sambrook, et al., MolecularCloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1985)). Conventional methods for preparation of cDNA frommRNA can also be used.

The particular cells or tissue from which the nucleic acids are isolatedwill depend upon the particular application. Typically, for detection ofabnormalities associated with cancer, genomic DNA is isolated from tumorcells. For prenatal detection of disease, fetal tissue will be used.

If the tissue sample is small, so that a small amount of nucleic acidsis available, amplification techniques such as the polymerase chainreaction (PCR) using degenerate primers can be used. For a generaldescription of PCR, see, PCR Protocols, Innis et al. eds. AcademicPress, 1990. In addition, PCR can be used to selectively amplifysequences between high copy repetitive sequences. These methods useprimers complementary to highly repetitive interspersed sequences (e.g.,Alu) to selectively amplify sequences that are between two members ofthe Alu family (see, Nelson et al., Proc. Natl. Acad. Sci. USA 86:6686(1989)).

CGH, at the cytogenetic level, facilitates the search for disease genesby identifying regions of differences in copy number between a normaland tumor genome, for example. For instance, CGH studies have beenapplied to the analysis of copy number variation in breast cancer (see,e.g., Kallioniemi et al. Proc. Natl. Acad. Sci. USA 91:2156-2160(1994)).

In CGH, the resolution with which a copy number change can be mapped ison the order of several megabases. With the present invention theresolution is a function of the length of the genomic DNA segmentscomprising the target nucleic acid sequences and the difference in mapposition between neighboring clones. Resolution of more than a factor of10 better than with standard CGH can be achieved with the presentinvention. This improved localization will facilitate efforts toidentify the critical genes involved in a disease, and permit moresensitive detection of abnormalities involving a small region of thegenome, such as in microdeletion syndromes.

Labeling nucleic acid probes

As noted above, the nucleic acids which are hybridized to the targetnucleic acids are preferably labeled to allow detection of hybridizationcomplexes. The nucleic acid probes used in the hybridization describedbelow may be detectably labeled prior to the hybridization reaction.Alternatively, a detectable label may be selected which binds to thehybridization product. As noted above, the target nucleic acid array ishybridized to two or more probe nucleic acids, either simultaneously orserially. Thus, the probes are each preferably labeled with a separateand distinguishable label.

The particular label or detectable group attached to the probe nucleicacids is selected so as to not significantly interfere with thehybridization of the probe to the target sequence. The detectable groupcan be any material having a detectable physical or chemical property.Such detectable labels have been well-developed in the field of nucleicacid hybridizations and in general most any label useful in such methodscan be applied to the present invention. Thus a label is any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means.

However, preferred labels produce an optical signal. Thus, particularlyuseful labels in the present invention include fluorescent dyes (e.g.,fluorescein isothiocyanate, texas red, rhodamine, and the like) andlabels that produce a colorimetric signal such as various enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA).

The nucleic acids can be indirectly labeled using ligands for whichdetectable anti-ligands are available. For example, biotinylated nucleicacids can be detected using labeled avidin or streptavidin according totechniques well known in the art. In addition, antigenic or haptenicmolecules can be detected using labeled antisera or monoclonalantibodies. For example, N-acetoxy-N-2-acetylaminofluorene-labelled ordigoxigenin-labelled probes can be detected using antibodiesspecifically immunoreactive with these compounds (e.g., FITC-labeledsheep anti-digoxigenin antibody (Boehringer Mannheim)). In addition,labeled antibodies to thymidine-thymidine dimers can be used (Nakane etal. ACTA Histochem. Cytochem. 20:229 (1987)).

Generally, labels which are detectable in as low a copy number aspossible, thereby maximizing the sensitivity of the assay, and yet bedetectable above any background signal are preferred. A label ispreferably chosen that provides a localized signal, thereby providingspatial resolution of the signal from each target element.

The labels may be coupled to the DNA in a variety of means known tothose of skill in the art. In a preferred embodiment the probe will belabeled using nick translation or random primer extension (Rigby, et al.J. Mol. Biol., 113: 237 (1977) or Sambrook, et al., Molecular Cloning--ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1985)).

Hybridization of labeled nucleic acids to targets

The copy number of particular nucleic acid sequences in two probes arecompared by hybridizing the probes to one or more target nucleic acidarrays (biosensors). The hybridization signal intensity, and the ratioof intensities, produced by the probes on each of the target elements isdetermined. Typically the greater the ratio of the signal intensities ona target nucleic acid the greater the copy number ratio of sequences inthe two probes that bind to that target sequence. Thus comparison of thesignal intensity ratios among target elements permits comparison of copynumber ratios of different sequences in the probes.

Standard hybridization techniques are used to probe a target nucleicacid array. Suitable methods are described in references describing CGHtechniques (Kallioniemi et al., Science 258: 818-821 (1992) and WO93/18186). Several guides to general techniques are available, e.g.,Tijssen, Hybridization with Nucleic Acid Probes, Parts I and II(Elsevier, Amsterdam 1993). For a description of techniques suitable forin situ hybridizations see, Gall et al. Meth. Enzymol., 21:470-480(1981) and Angerer et al. in Genetic Engineering: Principles and MethodsSetlow and Hollaender, Eds. Vol 7, pgs 43-65 (plenum Press, New York1985).

Generally, nucleic acid hybridizations utilizing the biosensors of thepresent invention comprise the following major steps: (1)prehybridization treatment to increase accessibility of target DNA, andto reduce nonspecific binding; (2) hybridization of the mixture ofnucleic acids to the nucleic acid targets on the biosensor; (3)posthybridization washes to remove nucleic acid fragments not bound inthe hybridization and (4) detection of the hybridized nucleic acidfragments. The reagent used in each of these steps and their conditionsfor use vary depending on the particular application.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. A number of methods for removing and/ordisabling the hybridization capacity of repetitive sequences are known(see, e.g., WO 93/18186).

For instance, bulk procedures can be used. In many genomes, includingthe human genome, a major portion of shared repetitive DNA is containedwithin a few families of highly repeated sequences such as Alu. Thesemethods exploit the fact that hybridization rate of complementarysequences increases as their concentration increases. Thus, repetitivesequences, which are generally present at high concentration will becomedouble stranded more rapidly than others following denaturation andincubation under hybridization conditions. The double stranded nucleicacids are then removed and the remainder used in hybridizations. Methodsof separating single from double stranded sequences include usinghydroxyapatite or immobilized complementary nucleic acids attached to asolid support. Alternatively, the partially hybridized mixture can beused and the double stranded sequences will be unable to hybridize tothe target.

Alternatively, unlabeled sequences which are complementary to thesequences whose hybridization capacity is to be inhibited can be addedto the hybridization mixture. This method can be used to inhibithybridization of repetitive sequences as well as other sequences. Forinstance, "Cot-1" DNA can be used to selectively inhibit hybridizationof repetitive sequences in a sample. To prepare Cot-1 DNA, DNA isextracted, sheared, denatured and renatured to a C₀ t˜1 (for descriptionof reassociation kinetics and C₀ t values, see, Tijssen, supra at pp48-54). Because highly repetitive sequences reanneal more quickly, theresulting hybrids are highly enriched for these sequences. The remainingsingle stranded (i.e., single copy sequences) is digested with S1nuclease and the double stranded Cot-1 DNA is purified and used to blockhybridization of repetitive sequences in a sample. Although Cot-1 DNAcan be prepared as described above, it is also commercially available(BRL). Reassociation to large C₀ t values will result in blocking DNAcontaining repetitive sequences that are present at lower copy number.

Analysis of detectable signals from hybridizations

Standard methods for detection and analysis of signals generated bylabeled probes can be used. In particular, the optical signal producedby binding of a labeled probe to a particular binding partner will becarried along the optical fibers 10, to which that binding partner isattached. As indicated above, the optical signal may be visualizeddirectly or transduced into an analog or digital electronic signal bymeans of a detector 20. To facilitate the display of results and toimprove the sensitivity of detecting small differences in fluorescenceintensity, a detector and a digital signal analysis system is preferablyused. The detector may be equipped with one or more filters to pass theemission wavelengths while filtering out excitation wavelengths therebyincreasing the signal to noise ratio. The use of filters will alsofacilitate distinguishing between binding events involving the two, ormore, differently labeled probes. Such detector/filter/signal processingsystems are well known to those of skill in the art.

What is claimed is:
 1. A method for comparing relative copy number ofnucleic acid sequences in two or more collections of nucleic acidmolecules, said method comprising:(a) providing a biosensor wherein thebiosensor comprises a plurality of optical fibers, each fiber includinga sensor end and a transmission end where the sensor ends of the opticalfibers bear target nucleic acids such hat the sensor end of at least onefirst fiber has attached a first target nucleic acid comprising a firsttarget nucleotide sequence, and the sensor end of at least one secondfiber has attached a second target nucleic acid, wherein said first andsecond target nucleic acids are different and are uniquely addressed;(b) contacting said biosensor with(i) a first collection of labellednucleic acid molecules, and (ii) at least a second collection oflabelled nucleic acid molecules; wherein the first and second labels aredistinguishable from each other; and (c) comparing the amount ofbinding, if any, of the first and second collections of labelled nucleicacid molecules to said first target nucleic acid, thereby determiningthe relative copy number of any sequences in the first and secondcollections that are substantially complementary to said first targetnucleotide sequence.
 2. The method of claim 1, wherein the targetnucleic acids are DNA.
 3. The method of claim 1, wherein the targetnucleic acids are cDNA.
 4. The method of claim 1, wherein the targetnucleic acids are RNA.
 5. The method of claim 1, wherein the targetnucleic acids are mapped to specific regions in human chromosomes. 6.The method of claim 1, wherein the target nucleic acids are about 400 toabout 1,000,000 nucleotides in complexity.
 7. The method of claim 1,wherein the complexity of any sequence substantially complementary tothe first or second target nucleotide sequences is less than 1% of thetotal complexity of the sequences in the sample.
 8. The method of claim1, wherein the first and second labels are fluorescent labels.
 9. Themethod of claim 1, wherein the first collection of labeled nucleic acidmolecules comprises mRNA or cDNA from a test cell and the secondcollection of labeled nucleic acid molecules comprises mRNA or cDNA froma reference cell.
 10. The method of claim 1, wherein the firstcollection of labeled nucleic acid molecules is from a test genome andthe second collection of labeled nucleic acid molecules is from a normalreference genome.
 11. The method of claim 10, wherein the test genomecomprises nucleic acid molecules from fetal tissue.
 12. The method ofclaim 10, wherein the test genome comprises nucleic acid molecules froma tumor.
 13. The method of claim 1, wherein the first and secondcollections of labeled nucleic acid molecules are treated to inhibit thebinding of repetitive sequences.
 14. The method of claim 13, wherein thefirst and second collections of labeled nucleic acid molecules are mixedwith unlabeled blocking nucleic acids comprising repetitive sequences.15. The method of claim 1, wherein said second target nucleic acidcomprises a second target nucleotide sequence, said method additionallycomprising comparing the amount of binding, if any, of the first andsecond collections of labeled nucleic acid molecules to said secondtarget nucleic acid, thereby determining the relative copy number of anysequences in the first and second collections that are substantiallycomplementary to said second target nucleotide sequence.
 16. The methodof claim 15, wherein said comparing step comprises determining the ratioof binding of the first and second collections of labeled nucleic acidmolecules to each target nucleic acid.
 17. The method of claim 16,wherein said comparing step additionally comprises comparing the ratiofor the first target nucleic acid to the ratio for the second targetnucleic acid.
 18. The method of claim 15, wherein said first or secondcollection of labelled nucleic acid molecules is selected from the groupconsisting of mRNA, cDNA, and genomic DNA.
 19. The method of claim 15wherein the sensor ends of the optical fibers are concave or convex. 20.The method of claim 15 wherein said biosensor comprises a plurality oftarget nucleic acids attached to said plurality of optical fibers toprovide a sensor face with a target nucleic acid concentration of about30,000 different target nucleic acids per square centimeter.
 21. Themethod of claim 15 wherein said biosensor comprises a plurality oftarget nucleic acids attached to said plurality of optical fibers toprovide a sensor face, wherein said sensor face is non-planar.
 22. Themethod of claim 15 wherein the amount of binding, if any, of the firstand second collections of labelled nucleic acid molecules is detectedusing a transilluminating light source.
 23. The method of claim 15,wherein said first or second target nucleic acid is indirectly attachedto the sensor end of said first or second fiber, respectively.